This invention relates to the use of the enzymes hexokinase and glucose-6-phosphate dehydrogenase in an analytical procedure for determining glucose.
In the determination of enzymes and other biological constituents, the reaction generally involves enzymes, coenzymes and substrates.
Enzymes are complex proteins with large molecular weights, and are usually of unknown chemical structure. They are classified by their substrate specificity, and catalytic activity. Enzymes are biological catalysts, which can catalyze the reaction of a single substrate, or the reaction of a group of similar substrates.
Coenzymes are organic chemicals with well-defined chemical structures. They usually have lower molecular weights than enzymes. They are required for specific enzyme assay or reaction. Coenzymes are detectably changed in their structure and/or atomic composition in the assay. Their reactions are stoichiometric with the substrate. With certain coenzymes having strong absorbence, the creation or disappearance of the absorbing form can be followed photometrically. For example, nicotinamide adenine dinucleotide (NAD) and reduced nicotinamide adenine dinucleotide (NADH) are used in many important clinical assays. Both species have a molecular weight of about 700. NADH absorbs strongly at 340 nm, while NAD does not.
Substrates are organic chemicals of known structure, whose reactions and interactions are catalyzed by enzymes resulting in a change in the substrate's chemical structure, atomic composition, or stereochemistry. In general, substrates are prone to degradation, both chemically and microbiologically. Substrates chemically degrade or hydrolyze in aqueous media, and serve as food for bacteria, fungi and other microorganisms. Typical substrates are glucose, lactate or lactic acid, gluconate and the like.
Because of their high specificity, the use of enzyme determinations has significantly increased during recent years. At present, the greatest limitation on the use of enzyme reagents lies in the unstable nature of the species therein. Numerous labile components are usually involved. To complicate matters, the exact nature of enzymes, as well as the mechanisms of their actions, remains unknown for the most part. Therefore, rigorous quality control measures are required to assure accurate and consistent results. Such measures can be costly.
In the prior art, to ensure strict quality control, emphasis was placed on stabilizing the labile ingredients in the reagents, i.e., to prevent them from degrading. For example, the enzyme or coenzyme can be locked into a solid matrix, either by dry blending, freeze drying, or by locking the chemical structure of the enzyme onto a solid matrix. These methods are expensive, require complicated manufacturing processes, and are less convenient for the user. Product uniformity is difficult to maintain with solid reagents. For example, many commercial freeze dried reference sera list an acceptable bottle-to-bottle variation of enzyme constituents at .+-.10% of the mean. More importantly, the user has to bear the burden of assuring the quality control in the dilution and use of the solid reagent. Because of these quality control problems with solid enzyme or coenzyme reagents, and because of the convenience factor, users generally prefer liquid, easy-to-use, and homogeneous reagents over solid (e.g. lyophilized) compositions.